Widening of the genetic and clinical spectrum of Lamb–Shaffer syndrome, a neurodevelopmental disorder due to SOX5 haploinsufficiency

Abstract

Lamb–Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved.
Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types of SOX5 alterations. Functional consequences of selected substitutions were investigated.
Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype–phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated.
This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

Full text

Widening of the genetic and clinical spectrum of
Lamb–Shaffer syndrome, a neurodevelopmental disorder
due to SOX5 haploinsufficiency
A full list of authors and affiliations appears at the end of the paper.
Purpose: Lamb–Shaffer syndrome (LAMSHF) is a neurodevelop-
mental disorder described in just over two dozen patients with
heterozygous genetic alterations involving SOX5, a gene encoding a
transcription factor regulating cell fate and differentiation in
neurogenesis and other discrete developmental processes. The
genetic alterations described so far are mainly microdeletions. The
present study was aimed at increasing our understanding of
LAMSHF, its clinical and genetic spectrum, and the pathophysio-
logical mechanisms involved.
Methods: Clinical and genetic data were collected through
GeneMatcher and clinical or genetic networks for 41 novel patients
harboring various types of SOX5 alterations. Functional conse-
quences of selected substitutions were investigated.
Results: Microdeletions and truncating variants occurred through-
out SOX5. In contrast, most missense variants clustered in the
pivotal SOX-specific high-mobility-group domain. The latter
variants prevented SOX5 from binding DNA and promoting
transactivation in vitro, whereas missense variants located outside
the high-mobility-group domain did not. Clinical manifestations
and severity varied among patients. No clear genotype–phenotype
correlations were found, except that missense variants outside the
high-mobility-group domain were generally better tolerated.
Conclusions: This study extends the clinical and genetic spectrum
associated with LAMSHF and consolidates evidence that SOX5
haploinsufficiency leads to variable degrees of intellectual disability,
language delay, and other clinical features.
Genetics in Medicine (2019) https://doi.org/10.1038/s41436-019-
0657-0
Keywords: autism; developmental delay; intellectual disability;
epilepsy; missense variants.
INTRODUCTION
The SOX protein family is made of transcription factors
harboring a high-mobility-group (HMG) domain at least 50%
similar to that of SRY (encoded by the sex-determining region
on the Y chromosome).
1
This domain mediates DNA binding
and bending, nuclear trafficking, and protein–protein interac-
tions. The 20 SOX proteins existing in humans and other
mammals fall into eight groups (SOXA to SOXH) based on
sequence identity within and outside this domain.
2,3
Most have
been shown in animal models to play pivotal roles in
determining the lineage choice, differentiation program, and
survival capacity of discrete cell types, such that as a whole the
SOX family controls many crucial biological processes,
including sex determination, neurogenesis, and skeletogenesis.
1
In humans, pathogenic variants in half of the SOX genes were
shown to date to cause developmental disorders.
4
For example,
SRY variants cause XY sex reversal (MIM 400045 and
400046);
5
SOX9 variants cause campomelic dysplasia with or
without XY sex reversal (MIM 114290);
5
SOX18 variants cause
hypotrichosis–lymphedema–telangiectasia syndrome (MIM
607823 and 137940);
6
and SOX4 and SOX11 (MIM 615866)
variants cause Coffin–Siris syndrome–like syndromes.
7,8
Most
pathogenic variants are de novo and, except for SRY,resultin
dominant disorders because of gene haploinsufficiency.
Lamb–Shaffer syndrome (LAMSHF, MIM 616803) was
initially described as a condition caused by de novo deletions
ranging from a few kilobases to several megabases and
including at least part of SOX5.
9
LAMSHF is clinically
characterized by developmental delays, language and motor
deficits, intellectual disability, behavioral disturbances includ-
ing autistic traits, and other, partially penetrant features.
9–12
SOX5 is located on chromosome 12p12.1 and gives rise to at
least five transcript isoforms through expression from
different promoters, alternative start site usage, and alter-
native precursor messenger RNA (pre-mRNA) splicing. The
longest isoform (NM_006940) encodes a 763–amino acid
Submitted 5 June 2019; revised 6 September 2019; accepted: 10 September 2019
Correspondence: Véronique Lefebvre (lefebvrev1@email.chop.edu) or Christel Depienne (christel.depienne@uni-due.de)
These authors contributed equally: Ash Zawerton, Cyril Mignot, Ashley Sigafoos
co-first authors: Ash Zawerton, Cyril Mignot, Ashley Sigafoos
Joint senior authors: Véronique Lefebvre, Karl J. Clark, Christel Depienne
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protein (originally referred to as L-SOX5, but more recently
and henceforward called SOX5) and is the predominant brain
isoform.
13
The shortest isoform (NM_178010) encodes a
protein corresponding to the L-SOX5 C-terminal half and is
testis-specific. All long protein isoforms contain the same
functional domains and are collectively critical in mouse
development.
14
Sox5
-/-
mice are born with lethal skeletal
malformations and with defective deep-layer cortical projec-
tion neurons, while Sox5
+/-
mice have a normal lifespan and
no obvious abnormalities.
15–18
To date, only a few SOX5 point variants, mostly introducing
premature termination codons, have been reported in
LAMSHF patients
19–21
or in large genetic studies of develop-
mental disorders without detailed clinical descriptions.
22–25
In
this study, we describe 41 unpublished patients carrying
various SOX5 deletions and point variants, including 16 with
missense variants. We delineate more precisely the clinical
spectrum associated with SOX5 alterations, aim at establishing
genotype–phenotype correlations, and explore pathogenicity
of selected variants using both in silico and functional
approaches.
MATERIALS AND METHODS
Human subjects
We collected clinical and molecular data from patients with
SOX5 microdeletions or point variants through Gene-
Matcher,
26
DECIPHER
27
(patient IDs 333039, 340665,
271393, 264625), and clinical networks. Referring physicians
used standard developmental scales and filled out a table with
detailed developmental, neurological, and behavioral history,
including imaging and electroencephalogram (EEG) data
where available. The study was approved by INSERM (RBM
C12-06). We obtained informed written consent for all
genetic studies as well as for the use of photographs shown in
Fig. 2g.
Genetic studies
Diagnostic laboratories performed genetic tests on blood
samples using microarrays or next-generation sequencing
(Supplementary Table 1). SOX5 variants and deletions were
validated and searched for in parents using Sanger sequencing
and fluorescence in situ hybridization (FISH) or real-time
polymerase chain reaction (PCR), respectively. SOX5 variants
were described based on the longest isoform (NM_006940.5)
using Alamut 2.11 (Interactive Biosoftware, France) and
Human Genome Variation Society guidelines (www.hgvs.
org/mutnomen). The InterVar interface was used to classify
SOX5 variants with adjusted criteria according to American
College of Medical Genetics and Genomics (ACMG)
recommendations.
28,29
Combined annotation dependent
depletion (CADD) scores
30
were calculated for each variant
(Supplementary Table 1). SOX5 isoforms and promoters and
other SOX sequences were retrieved from the National
Center for Biotechnology Information (NCBI) and Fantom5
databases and sequences were aligned using ClustalW
(MacVector16 software). The effects of missense variants on
protein structure and function were predicted using HOPE
31
and Swiss-Model.
32
SOX5 variants were queried in human
populations using gnomAD. Data were statistically analyzed
using Fisher’s exact and Wilcoxon–Mann–Whitney tests.
SOX5 plasmids
Expression plasmids for the longest SOX5 isoform and
variants thereof were generated in the pKTol2C-EGFP
plasmid.
33
The EGFP sequence was replaced with custom-
synthesized or PCR-amplified SOX5 sequences (primers are
available upon request). Plasmid integrity was verified using
Sanger sequencing.
SOX5 immunolocalization
HEK-293 cells (ATCC
®
CRL-1573
™
)wereplatedonglass
coverslips and transfected with pKTol2C-SOX5 plasmids (2 μg)
and Lipofectamine 2000 Transfection Reagent (Thermo Fisher
Scientific). Two days later, they were stained using Image-IT
TM
LIVE Plasma Membrane and Nuclear Labeling Kit (Thermo
Fisher, I34406), fixed in 4% paraformaldehyde, permeabilized
with 0.1% Triton X-100 in PBS (PBST), and blocked in PBST
supplemented with 1% BSA and 22.5 mg/ml glycine. They were
then incubated with rabbit polyclonal SOX5 antibody (1:200,
Abcam, ab94396), followed by goat anti-rabbit antibody (1:500,
Alexa Fluor 488, Invitrogen, A27034). After placing DAPI-
containing Vectashield antifade mounting medium (Vector
Laboratories), cells were imaged by confocal laser scanning
microscopy (Zeiss LSM 780; 100× objective).
Western blot, electrophoretic mobility shift, and
dimerization assays
HEK-293 cells were plated in six-well dishes and transfected
eight hours later with empty or SOX5 expression plasmid (1
µg) and FuGENE6 (3 μl, Promega). The next day, extracts
were prepared using NE-PER Nuclear and Cytoplasmic
Extraction Reagents (Thermo Fisher Scientific) and tested
by western blotting using SOX5 antibody (1:1000) and
horseradish peroxidase–conjugated goat anti-rabbit IgG
(1:5000, Vector Biolabs). Signals were visualized using ECL
Prime Western Blotting Detection Reagents (Amersham).
Electrophoretic mobility shift assay (EMSA) was conducted
using the same extracts, 10 fmoles [α-
32
P]-dCTP-labeled
2HMG probe and 1 μg poly(dG-dC).poly(dG-dC), as
described.
34
Homodimerization was tested in western blots
following cell extract incubation for 10 minutes with 0.01%
glutaraldehyde.
Reporter assay
HEK-293 cells were transfected with FuGENE6 containing
150 ng pSV2βGal, 500 ng Acan [4xA1]-p89Luc reporter, 50
ng SOX9 expression plasmid, and 300 ng plasmid encoding
no protein, wild-type (WT) SOX5, and/or variant SOX5, as
previously described.
35
Forty hours later, cells were collected
in Tropix Lysis buffer (Applied Biosystems) with protease
inhibitor cocktail (Thermo Fisher Scientific) and tested using
Dual-Light luciferase and E. coli β-galactosidase assays
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(Thermo Fisher Scientific). Reporter activities were calculated
as means with standard deviation of luciferase values
measured for triplicates and normalized for transfection
efficiency using β-galactosidase values.
RESULTS
The SOX5 variant spectrum associated with LAMSHF
includes missense variants
We collected genetic and clinical information from 41
patients (Table 1, supplementary table 1). Eight patients
(D1–D8), representing seven families, carried novel patho-
genic microdeletions. These microdeletions ranged from 43.7
kb to 1.7 Mb and involved different breakpoints (Fig. 1a).
While the largest deletion encompassed the entire SOX5 gene
and its 5’neighbor (BCAT1), the others were restricted to
various segments of SOX5.
The other 33 patients belonged to 31 families and totaled 23
distinct point variants. Nineteen of these variants were
classified by the InterVar interface as pathogenic or likely
pathogenic (P1–P29) and the other four as variants of
unknown significance (VUS) (V1–V4). Two patients had
indels introducing frameshifts (P7 and P10) (Table 1, Fig. 1a,
b). Two (P13 and P14) had variants altering the acceptor and
donor splice sites of the coding exon 12, respectively. Thirteen
patients (including a pair of dizygotic twins) totaled eight
distinct nonsense variants (P1–P6, P8–P10, P11, P12, P15,
P25, and P28). Truncating variants (i.e., nonsense, splice site,
and frameshift variants) were scattered over the L-SOX5
isoform from the N-terminus to the middle of the HMG
domain. All truncating SOX5 variants thus encode proteins
lacking DNA-binding ability. Furthermore, since all variants
spare the last exon, they likely trigger nonsense-mediated
mRNA decay and thus prevent protein expression. Sixteen
patients (including a sib pair) had 11 different missense
variants (P16–P24, P26, P27, P29, and V1–V4). Seven of these
variants were clustered in the HMG domain, while the four
VUS occurred in the first coiled coil (V1), between the coiled
coils (V2), or after the HMG domain (V3 and V4).
Five identical nucleotide transitions were identified in several
unrelated individuals: c.622C>T, p.Gln208* (P2 and P3);
c.637C>T, p.Arg213* (P4/P5 and P6); c.1477C>T, p.Arg493*
(P11 and P12); c.1678A>G, p.Met560Val (P16 and P17); and
c.1711C>T, p.Arg571Trp (P19 to P23). Besides a few that were
of unknown inheritance, these alterations were all de novo and
thus suggested the presence of hot spots for nucleotide
transitions. Of additional note, 17 of 22 single-nucleotide
variants identified in patients are C>T and G>A transitions,
suggesting that many SOX5 point variants result from cytosine
deamination, a prevailing mechanism of genetic alteration.
36
High rate of parental mosaicism
Most microdeletions and variants predicted to be pathogenic
or likely pathogenic were undetected in parental blood
samples, suggesting de novo occurrence (25/34 families,
74%). However, in each of three families, the same alteration
was found in two affected siblings (D6 and D7; P4 and P5;
and P22 and P23), but not in their parents, and in two other
patients (D3 and P9), the variant was present at low levels in
maternal blood. In addition, one nonsense variant was
transmitted to a patient (P1) from his affected mother, where
it was de novo. Variant transmission could not be determined
for four patients (D1, P7, P12, P19) due to unavailability of
parental samples. These findings thus indicate that pathogenic
LAMSHF variants are frequently inherited from a mosaic
parent (5/34, 15%) and also occasionally from an affected
parent (1/34, 3%).
Wide clinical spectrum associated with SOX5 pathogenic
alterations
Excluding the four patients with VUS, our patient series
comprised 20 females and 17 males (Supplementary Table 1).
The patients were 12.2 years of age on average at the time of
examination (median: 8.0 years, range: 1.75–36), with 11 older
than 15 and six younger than 4.
For most patients, pregnancy and delivery were unremark-
able (21/36), birth measurements (weight, length, and head
circumference) normal (15/18 for whom full information was
available), and the neonatal period uneventful (25/36). Eight
patients had mild growth retardation or a small head at birth,
two were hypotonic, and three had feeding difficulties.
Developmental delay was present in all patients for whom
information was available. Although more than half of the
patients timely acquired the sitting position (≤9 months; n=
16/29), the age of walking was delayed in all but one
(>18 months; n=35/36), without clear timing differences
among variant categories (Fig. 2a, b). The age of first words
was delayed in 21/26 patients (>12 months; mean:
29.9 months, range: 10–60 months). The delay was sig-
nificantly less pronounced in patients with missense variants
(mean: 22.4 months, n=11) than in those with deletions and
truncating variants (mean: 35.2 months, n=15; pvalue: 0.04,
Wilcoxon rank sum test) (Fig. 2c). The levels of verbal
expression were variable, but most patients older than three
years could make short or full sentences (Supplementary
Tables 1 and 2).
Intellectual disability (ID) was reported in 30/33 patients,
with 27 having mild-to-moderate ID and 3 having severe or
moderate-to-severe ID. The three patients without ID had
learning difficulties and either borderline functioning or
discrepant verbal/performance IQ scores. No significant
correlation was observed between degree of ID and variant
type (Fig. 2d).
Of 25 patients evaluated for autism spectrum disorder
(ASD), 6 (4 with truncating variants and 2 with missense
variants) were positively diagnosed (24%) and 11 had other
behavioral disturbances including stereotypies, isolation,
tantrums, and hyperactivity (Fig. 2e). Of 36 patients, 8
experienced epileptic seizures (22%), but 5 of these had only
one or two episodes and did not require medication. One of
these patients (D6) had seizures triggered by environmental
photosensitivity, an unusual finding in a “developmental
delay plus seizures”syndrome (Supplementary Fig. 1).No
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Table 1 Summary of genetic and clinical data
ID Family
history
Age
(years)
Sex SOX5 variant
(NM_006940)
Variant type Inheritance ACMG class Language delay Language
ability
ID (level) Behavioral
disturbances
Seizures Other features
D1 Spo 6.5 M Del ex1 Del Unknown 5 Y 10 words Y (mild) NA N Hypotonia
D2 Spo 10 M Del ex5–10 Del DN 5 Y NA Y (moderate) Intolerant to
frustration,
heteroaggressive
N Strabismus, hypotonia, fused vertebrae
D3 Spo 14 F Del ex6–15 Del Mat mosaic 5 Y Sentences Y (mild) NA N Strabismus
D4 Spo 5.5 F Whole-gene del
(+BCAT1)
Del DN 5 Y NA Y (moderate) NA N Hypotonia
D5 Spo 10 F Del ex6–8 Del DN 5 Y Short sentences Y (moderate) NA N Strabismus
D6 Sib 26 F Del ex8–10 Del Mosaic 5 NA NA NA N Y Fused vertebrae, subaortic ventricular septal
defect, pulmonary stenosis
D7 Sib 36 M Del ex8–10 Del Mosaic 5 Y Sentences N (dyspraxia) NA Y None
D8 Spo 24 M Del ex10–15 Del DN 5 Y Sentences Y (moderate) Autistic features,
blinking, phobia
N Vesicoureteral reflux, strabismus, amblyopia,
pyramidal syndrome, toe syndactyly
P1 Mat 2.5 M c.518G>A,
p.(Trp173*)
NS Mat (DN in
mother)
5 Y 20 words Y (mild–moderate) Stereotypies,
intolerant to
frustration
N Strabismus, hypotonia
P2 Spo 7 F c.622C>T,
p.(Gln208*)
NS DN 5 Y Words Y (mild) NA N None
P3 Spo 3 F c.622C>T,
p.(Gln208*)
NS DN 5 Y Words NA NA NA Hypotonia, café au lait spots
P4 Sib 31 F c.637C>T,
p.(Arg213*)
NS Mosaic 5 Y Sentences Y (mild) Isolated, shy, anxious N Strabismus, optic atrophy, saccadic pursuit,
tetrapyramidal syndrome, scoliosis
P5 Sib 31 M c.637C>T,
p.(Arg213*)
NS Mosaic 5 Y Sentences Y (mild) Aggressive, agitated N Saccadic pursuit, tetrapyramidal syndrome,
scoliosis
P6 Spo 17.8 F c.637C>T,
p.(Arg213*)
NS DN 5 Y Sentences N (borderline IQ) N N Strabismus, myopia, optic atrophy, hypotonia,
gait instability, dysmetria
P7 Spo 2.5 F c.747_748del,
p.(Arg250Thrfs*36)
FS Unknown 5 Y Nonverbal NA NA N Strabismus, hypotonia
P8 Spo 8 M c.820C>T,
p.(Gln274*)
NS DN 5 Y Short sentences Y (mild–moderate) ASD, intolerant to
frustration
N Strabismus, hypotonia, ear tubes, undescended
testicles
P9 Spo 7 F NS Mat mosaic 5 Y Associates words Y (mild–moderate) ADHD N Feeding difficulties, hypotonia
P10 Spo 8 M c.1465dup,
p.(Leu489Profs*3)
FS DN 5 Y Words NA ASD N None
P11 Spo 3 F c.1477C>T,
p.(Arg493*)
NS DN 5 Y Words Y (moderate) NA N Hypotonia, cortical visual impairment, ear tubes,
scoliosis, constipation, supernumerary nipple
P12 Spo 20 M c.1477C>T,
p.(Arg493*)
NS Unknown 5 Y Nonverbal Y (severe) NA Y Scoliosis
P13 Spo 12 F c.1489-1G>A, p.? Splice DN 5 Y Sentences Y (moderate) ASD, anxious N Hypotonia, constipation, thoracic kyphosis
P14 Spo 8 F c.1597+2T>A, p.? Splice DN 5 Y >100 words Y (moderate) NA N (EEG
anomalies)
Hypotonia, right microtia, microcephaly, myopia
P15 Spo 30 M c.1613C>G,
p.(Ser538*)
NS DN 5 Y Short sentences Y (moderate) Autistic features Y None
P16 Pat 13 M c.1678A>G,
p.(Met560Val)
MS DN 5 Y Sentences Y (mild) Temper tantrums Y None
P17 Spo 1.75 F c.1678A>G,
p.(Met560Val)
MS DN 5 Y Words Y (mild) NA N Hypotonia, joint hyperlaxity, hip dysplasia
P18 Sib 4 M c.1681A>C,
p.(Asn561His)
MS DN 5 Y Words Y (severe) Insomnia N Dysphagia with G-tube dependence, scoliosis,
hypotonia, muscle weakness
P19 Spo 27 F c.1711C>T,
p.(Arg571Trp)
MS Unknown 5 Y Sentences Y (mild) NA N Ovarian dystrophy
P20 Pat 17 M c.1711C>T,
p.(Arg571Trp)
MS DN 5 Y Sentences Y (moderate) Autistic features N None
P21 Mat 6 M c.1711C>T,
p.(Arg571Trp)
MS DN 5 Y Short sentences Y (moderate) Angry outbursts,
stereotypies
Y Joint hyperlaxity, strabismus, hypotonia
P22 Sib 3.3 F c.1711C>T,
p.(Arg571Trp)
MS Mosaic 5 Y Words Y (mild–moderate) NA N Hypotonia
P23 Sib 5.6 M c.1711C>T,
p.(Arg571Trp)
MS Mosaic 5 Y Short sentences Y (mild–moderate) ADHD N Hypotonia, microcephaly
P24 Spo 18 F c.1712G>T,
p.(Arg571Leu)
MS DN 4 Y Sentences Y (moderate) NA Y Hypotonia
P25 Spo 4 F c.1782G>A,
p.(Trp594*)
NS DN 5 Y Babbles Y (moderate–severe) ASD, sensory
integration disorder
N Bilateral optic nerve atrophy, hypotonia
P26 Spo 14 F c.1786G>C,
p. (Ala596Pro)
MS DN 5 Y Sentences Y (moderate) ASD, anxious Y Teeth anomalies, strabismus, hypotonia, muscle
weakness
P27 Spo 6.5 F c.1814A>G,
p.(Tyr605Cys)
MS DN 5 Y Sentences Y (borderline–mild) ASD N Constipation, optic atrophy, joint hyperlaxity,
hypotonia
P28 Spo 5.5 M c.1819G>T,
p.(Glu607*)
NS DN 5 Y Sentences N (VIQ/PIQ
discrepancy)
NA N Ear tubes, pallor of left optic nerve, strabismus,
constipation, hypotonia
P29 Spo 4 M c.1868A>G,
p.(Tyr623Cys)
MS DN 4 Y Words Y (mild) NA N Gait ataxia
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correlation was found between the occurrence of seizures and
the SOX5 variant type (Fig. 2f).
Clinical examination revealed that stature and weight were
within normal range for most patients. Head circumference of
both males (n=14) and females (n=15) was in the low but
normal range (~−1.5 SD) while two patients (P14 and P23) had
microcephaly. Hypotonia was reported in 22 patients, and five
had additional neurological features, including ataxia (n=2) or
pyramidal syndrome (n=3). Thirty-one patients had mild
dysmorphic facial features, including broad/full nasal tip (n=
9), thin upper lip or full lips (n=8),smalljaworchin(n=5),
long face (n=3), or epicanthus (n=3). Strabismus was
reported in 13 patients, optic atrophy in 5, and amblyopia or
cortical visual impairment in 1 each. Except for thin optic
nerves, brain magnetic resonance image (MRI) scans were
normal or showed nonspecific anomalies. Besides dysmorphic
facial features, other skeletal malformations included scoliosis in
six patients, thoracic kyphosis and hip dysplasia in one patient
each, and fused cervical vertebrae in two patients (Supplemen-
tary Table 1). Malformations of other organs were rare and
restricted to individual patients. Again, no correlation was
found between the occurrence of these features and the variant
types. Moreover, patients with recurrent variants (e.g., P2–P3: p.
Gln208*, P4-P6: p.Arg213*, P16–P17: p.Met560Val, and
P19–P23: p.Arg571Trp) exhibited considerable clinical varia-
bility, indicating that factors other than the SOX5 variants
modulate the expression of the clinical phenotype.
SOX5 is tightly conserved in the general population
We used gnomAD, a genomic database for over 140,000
individuals who are theoretically unrelated and lacking severe
pediatric disease, to investigate conservation constraints on
SOX5 in humans.
37
While 158 synonymous variants were
predicted and 159 were observed (Z- score: −0.08), 42 loss-of-
function variants were expected, but only 3 were observed
(probability of loss-of-function intolerance [pLI] =1). More-
over, 427 missense variants were predicted, but only 244 were
observed (Z-score:3.21).Thus,SOX5 is under tight conserva-
tion constraint in control populations. Interestingly, gnomAD
synonymous variants were found for 10–29% residues both
within and outside functional domains, whereas missense
variants altered significantly fewer residues in the HMG domain
(six residues, i.e., 7.5%) than in other regions (21–33%) and
significantly fewer than synonymous variants (20%, p=0.017)
(Fig. 3a, b). The SOX5 HMG domain is thus highly constrained
within control populations, which is in contrast to the relatively
high prevalence of HMG domain missense variants observed in
our patient cohort. The first coiled-coil domain also had
significantly fewer missense variants (20.7%) than the regions
outside of known functional domains (33.2%; p=0.03),
suggesting that this domain, which is required for SOX5
homodimerization and thereby for binding to pairs of
recognition sites in target genes, is also under conservation
constraint.
The six HMG domain missense variants found in gnomAD
affected two of the same residues as in LAMSHF patients and
Table 1 continued
ID Family
history
Age
(years)
Sex SOX5 variant
(NM_006940)
Variant type Inheritance ACMG class Language delay Language
ability
ID (level) Behavioral
disturbances
Seizures Other features
V1 Spo 18 M c.703C>T,
p.(Arg235Cys)
MS DN 3 NA NA N NA N Tourette syndrome
V2 Spo 0.7 M c.928T>A,
p.(Cys310Ser)
MS DN 3 NA NA NA NA Y Severe obstructive sleep apnea, severe
gastroesophageal reflux, G-tube fed, hypotonia
V3 Spo 13 M c.1895C>A,
p.(Thr632Asn)
MS DN 3 Y Words Y (moderate–severe) ASD N None
V4 Spo 36 M c.2078C>T,
p.(Ser693Leu)
MS DN 3 Y Short sentences Y (moderate) NA Y None
IDs: D, pathogenic microdeletion; P, pathogenic or likely pathogenic variant; V, variant of unknown significance. American College of Medical Genetics and Genomics (ACMG) class: 5, pathogenic; 4, likely pathogenic; 3,
variant of uncertain significance. See Supplementary Table 1 for more details.
ADHD attention deficit–hyperactivity disorder, ASD autism spectrum disorder, Del deletion, DN de novo, EEG electroencephalogram, ex exon(s), Ffemale, FS frameshift, G-tube gastric tube, Mmale, Mat maternal, MS mis-
sense, Nno, NA not available, NS nonsense, Pat paternal, PIQ performance IQ, Sib sibling (affected sib pairs), Spo sporadic, VIQ verbal IQ, Yyes.
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four others, and all six occurred only once (Supplementary
Table 5). In contrast, for missense variants located outside the
HMG domain, we found four occurrences of the patient
Arg235Cys variant (located in the first coiled coil) in
gnomAD, and one for Ser693Leu. Other gnomAD variants
affected the same residues as in patients, such as Arg235His,
found in 11 individuals. These observations suggest that some
SOX5 variants, especially those located outside the HMG
domain, may be better tolerated than others.
In silico prediction of variant pathogenicity
To predict pathogenicity of SOX5 missense variants, we first
examined the location and conservation of affected residues.
SinceallHMGdomainresiduesarefullyorsemiconserved
in SOX5 vertebrate orthologs (Supplementary Fig. 2a), we
focused on human SOX protein paralogs. All HMG domain
residues altered in patients and gnomAD individuals
affected residues involved in DNA binding or bending,
α-helical configuration, or nuclear trafficking (Fig. 3c).
Interestingly, 3 of the 5 residues altered in patients (Met560,
Asn561 and Arg571) were among 23 residues identical in all
protein paralogs, Tyr605 was among 13 semiconserved
residues, and only Ala596 was among the 40 nonconserved
residues. Conversely, only two of the six residues altered in
gnomAD individuals were among the conserved and
semiconserved ones. Outside the HMG domain, patient
variants affected residues that are highly conserved in SOX5
and its orthologs (Supplementary Fig. 2b). When the
comparison was limited to human SOXD proteins (SOX5,
SOX6, and SOX13), these conservation patterns held
strongly for Arg235Cys, located in the first coiled-coil
domain, and Thr632Asn, immediately flanking the HMG
domain, but less strongly for residues located in functionally
unknown regions (Fig. 3d). Together, these data suggested
that all HMG domain variants and a few other patient
variants might impact SOX5 function.
We then asked whether the HMG domain residues altered
in LAMSHF patients also cause disease when altered in other
SOX genes. Interestingly, all residues affected in LAMSHF
patients were shown to cause gonadal dysgenesis or XY sex
reversal when altered in SRY, or campomelic dysplasia with or
without XY sex reversal when altered in SOX9 (Supplemen-
tary Table 6). In contrast, only two of the four variants found
in gnomAD, but not in LAMSHF patients, were shown to
D1
D4
D2
D5 D8
Missense variant in the HMG domain
Missense variant outside the HMG domain
Nonsense variant
Frameshift variant
Splice variant
D3
D6 & D7
p2 p8
p11
1 2 3 4 56789 1011121314 15
p1
p4
NM_006940.5
a
b
NM_001261414.2
NM_001261415.2
NM_152989.4
NM_178010.2
5′UTR
SOX5 (NM_006940.4)
1 100 193
1st CC 2nd CC HMG
200 274 448 493 555 630300 400 500 600 700 763
3′UTR1st CC
R213*
(2x)
Q274*
R235C C310S
R250fs*
E246fs*21 T499fs*21 A597fs*21
R611G22
G354*19
P302S25
L489fs*
R471*
R538*
W594*
M560V (2x)
N561H
R571L
A596P23
T632N24
S693L
Y605C
Y623C
R571W (4x)23
R493* (2x)
c.1489–1G>A
c.1597+2T>A
Q208*
(2x)
W173*
R18*19
c.741+1G>A*23
2nd CC HMG
p3
p5, p9, p10, p15
Fig. 1 SOX5 variant spectrum associated with Lamb–Shaffer syndrome (LAMSHF). (a) Location of genetic alterations identified in patients in this
study. SOX5 transcript isoforms are labeled with National Center for Biotechnology Information (NCBI) accession numbers. Boxes 1 to 15, coding exons of
isoform NM_006940. 5’and 3’UTR: 5’and 3’untranslated sequences. p1 to p11 represent SOX5 promoters listed in the Fantom5 database; p1 and p2 (in
bold) are the main promoters driving SOX5 expression in brain. CC, coiled-coil domain. Double-arrowed lines, deletions in patients D1–D8. Point variants,
labeled as indicated. (b) Location of point variants reported here (above) and previously (below) on the longest SOX5 isoform. Protein and domain residue
boundaries are indicated underneath the schematic. Red, nonsense and frameshift variants. Blue and green, missense variants within and outside the HMG
domain, respectively. Superscripts, references.
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cause disease when altered in SRY. These data further support
pathogenicity of patient variants. They also suggest that some
variants present in gnomAD individuals could be pathogenic,
but clinical information was unavailable to validate this
possibility.
Lastly, comparison of WT and variant residues using
HOPE (Supplementary Fig. 3) showed that all variants
differed from WT residues by at least one major structural
feature: 16/18 differed in size, 13/18 differed in hydro-
phobicity, and 6/6 had a neutral instead of positive charge.
gD2 D6 D8
P1
P13 P14 P25 P28
P1’s mother P6 P10
Age at sitting (months)
ab
c
def
Age at walking (months)
Age at first words (months)
Deletion Truncating
Number of patients with
“estimated ID level”
Number of patients with
“behavioral disturbances”
Number of patients with
“epilepsy”
Seizures No seizures
Normal-borderline
Mild
Moderate
Severe
4
3
2
1
0
4
5
3
2
1
0
12
14
10
6
8
4
0
2
Borderline-mild
None ASD Other
Mild-moderate
Moderate-severe
Missense Deletion Truncating Missense Deletion Truncating Missense
Deletion Truncating Missense Deletion Truncating Missense Deletion Truncating Missense
35
30
25
20
15
10
5
70
60
50
40
30
20
60
50
40
30
20
10
Fig. 2 Patients exhibit similar clinical features regardless of the SOX5 alteration type. Box plots showing comparative distribution of ages at (a)
sitting unsupported, (b) walking unsupported, and (c) first words for patients with deletion, truncating, and missense variants. (d) Number of patients with
normal to borderline cognitive abilities and various degrees of intellectual disability (ID). (e) Number of patients with autism spectrum disorder (ASD) or other
behavioral disturbances. (f) Number of patients with seizures. (g) Facial profiles of individuals with de novo SOX5 variants. Above: D2 at age 10 years; D6 at
age 26 years; D8 at age 24 years. Center: P1 at age 2 years, and his mother (41 years old); P6 at age 19 years; P10 at age 8 years. Below: P13 at ages 2 years,
6 months, and 11 years, 4 months, respectively; P14 at ages 2 years, 4 months and 8 years, respectively; P25 at age 4 years; P28 at age 5 years. Common
facial features include broad or full nasal tip, thin upper lip and/or full lower lips, small jaw or prominent chin, prominent upper incisors and epicanthus.
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1st CC 2nd CC HMG domain
1st CC 2nd CC HMG domain
104
a
b
c
d
103
Synonymous variants
Synonymous variants
Missense variants
Missense variants
102
10
8
6
4
2
0
104
103
102
10
8
6
4
2
0
50 0.58
0.16
0.03
0.12
0.28
19.6
29.3
10.0
20.0
0.00001
0.77 0.002
0.02
0.03
33.2
20.7
30.0
7.5
40
30
20
% residues with variant (s)
10
0
SRY
SOX1
SOX2
SOX3
SOX14
SOX21
SOX4
SOX11
SOX12
SOX5
SOX6
SOX13
SOX8
SOX9
SOX10
SOX7
SOX17
SOX18
SOX15
SOX30
H1 H2 H3
DNA binding
& bending
α-helices
NLS & NES
SOX5
SOX6
SOX13
50
40
30
20
% residues with variant (s)
10
0
Other
H555Y R571Q R593C A596S L600V P629S
M560V N561H R571L & W
R235C C310S S693L T632L
A596P A605C
1st CC 2nd CC HMG Other 1st CC 2nd CC HMG
SOX5 residues
Number of variant alleles per residue
1
9
17
25
33
41
49
57
65
73
81
89
97
105
113
121
129
137
145
153
161
169
177
185
193
201
209
217
225
233
241
249
257
265
273
281
289
297
305
313
321
329
337
345
353
361
369
377
385
393
401
409
417
425
433
441
449
457
465
473
481
489
497
505
513
521
529
537
545
553
561
569
577
585
593
601
609
617
625
633
641
649
657
665
673
681
689
697
705
713
721
729
737
745
753
761
1
9
17
25
33
41
49
57
65
73
81
89
97
105
113
121
129
137
145
153
161
169
177
185
193
201
209
217
225
233
241
249
257
265
273
281
289
297
305
313
321
329
337
345
353
361
369
377
385
393
401
409
417
425
433
441
449
457
465
473
481
489
497
505
513
521
529
537
545
553
561
569
577
585
593
601
609
617
625
633
641
649
657
665
673
681
689
697
705
713
721
729
737
745
753
761
Fig. 3 Human SOX5 is under tight conservation constraint. (a) Distribution of synonymous and missense variants in SOX5 in gnomAD individuals.
CC, coiled coil. (b) Percentages of residues carrying at least one synonymous or missense variant in the functional and other domains of SOX5 in gnomAD
individuals. T-tests were performed to calculate the statistical significance of differences between protein domains. Pvalues are indicated. (c) Alignment of all
human SOX protein HMG domain sequences, with indication of residues altered in Lamb–Shaffer syndrome (LAMSHF) patients (red) and altered only in gnomAD
individuals (purple). Asterisks, fully conserved residues. Dots, semiconserved residues. Colored triangles, residues important for DNA binding and bending.
Brackets, H1, H2, and H3 α-helices. Continued lines linked with dotted lines, key amino acids in nuclear localization signal sequences (NLS) and nuclear export
signal sequence (NES). (d) Alignment of human SOXD protein sequences outside the HMG domain that encompass residues altered in LAMSHF patients.
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All variants could thus affect the secondary structure and
hence function of SOX5.
Overall, these analyses concurred that most missense
variants identified in our patient series are likely pathogenic.
Truncating variants and missense variants located within or
near nuclear import signals impair SOX5 translocation to
the nucleus
We constructed expression plasmids for WT and variant
forms of L-SOX5 and transiently transfected them in HEK-
293 cells to explore the functional impacts of variants.
Western blots of nuclear and cytoplasmic fractions (Fig. 4a)
and cell immunostaining assays (Fig. 4b) showed that WT
SOX5 localized primarily in the nucleus, as expected. On the
contrary, expression of nonsense variants (Gln208*, Gln274*,
Gly354*, and Arg493*) revealed that, if these variants were
expressed in patients’cells (i.e., if their mRNAs were not
subjected to nonsense-mediated decay), they would be
primarily cytoplasmic. This result was expected since protein
truncation occurs before the nuclear translocation signals. All
proteins with a missense variant that we tested were able to
translocate into the nucleus, except those in which the variant
occurred within or near the N-terminal nuclear import signal.
Accordingly, the Met560Val variant was localized to both the
cytoplasm and nucleus, and the Asn561His and Arg571Trp
variants were mainly cytoplasmic. Cytoplasmic retention of
these missense variants may thus contribute to pathogenicity.
Missense variants in the HMG domain prevent SOX5 from
participating in transactivation
We tested the transcriptional activity of SOX5 variants by
transfecting HEK-293 cells with an Acan reporter whose
enhancer is synergistically activated by SOX9 and SOXD
proteins.
35
WT SOX5 increased transactivation by SOX9 in a
dose-dependent manner (Fig. 4c). Nonsense and HMG
domain missense variants exhibited little if any activity,
whereas missense variants located outside the HMG domain
had activity similar to WT. Since SOX5 variants are
heterozygous in our patients, we also tested whether they
could interfere with the activity of WT SOX5. Nonsense and
HMG domain missense variants did not affect the activity of
WT SOX5, and missense variants located outside the HMG
domain increased the reporter activity as much as WT SOX5
(Fig. 4d). Thus, none of the variants showed a dominant-
negative effect.
We then tested the DNA-binding ability of SOX5
missense variants in EMSA using whole-cell extracts from
HEK-293 cells transfected with SOX5 plasmids and a probe
avidly binding SOXD homodimers.
38
HMG domain mis-
sense variants failed to bind DNA, whereas other missense
variants efficiently bound DNA (Fig. 4e). This result also
suggested that Arg235Cys, located in the main coiled-coil
domain, can homodimerize effectively. Its ability to
homodimerize was confirmed in an assay where closely
interacting proteins were crosslinked with glutaraldehyde
(Fig. 4f).
In conclusion, HMG domain missense variants prevented
SOX5 from binding DNA and from participating in
transcriptional activation, supporting their pathogenicity.
On the contrary, variants located outside the HMG domain
had no deleterious impact in the assays used, but this finding
does not rule out that they could be pathogenic and alter
other, untested SOX5 activities.
DISCUSSION
LAMSHF syndrome was previously described in just over two
dozen patients. Most patients had deletions of at least part of
SOX5, and a few had either a chromosomal translocation
involving SOX5,orSOX5 nonsense or frameshift variants.
9–
12,19–21
Our patient series more than doubles the number of
cases described in the literature and demonstrates that SOX5
missense variants clustering in the HMG domain can also cause
LAMSHF syndrome. All variants were heterozygous, and most
were predicted in silico and validated in vitro to be loss-of-
function variants. This confirms that SOX5 haploinsufficiency is
deleterious for neurogenesis and a few other developmental
processes. Our study also revealed that parental mosaicism,
found in at least 14% of families in our series, is relatively
frequent in LAMSHF syndrome. This finding is important for
genetic counseling and in line with increasing evidence that
somatic, gonosomal, or gonadal mosaicism in parents may
cause recurrence of neurodevelopmental disorders, apparently
due to de novo variants.
39
SOX5 and LAMSHF syndrome thus
expand the list of such genes and disorders.
Our extended study allowed further definition of the
LAMSHF clinical features. ID is mostly within the mild-to-
moderate range, and some cases have specific cognitive
deficits rather than ID.
9
Delays in motor and language
acquisition are observed in all patients and correlate with the
level of ID. Behavioral disturbances are frequent and include
ASD or autistic traits, as previously reported.
9,10,40
Micro-
cephaly is infrequent; yet, brain growth seems frequently
mildly altered. Hypotonia is common, whereas other
neurological features are infrequent. Our findings also suggest
that SOX5 pathogenic variants predispose to epilepsy, with a
prevalence of an order of magnitude higher than in the
general population. Seizures in SOX5 patients usually respond
well to antiepileptic treatments and follow a benign course.
Ophthalmologic features, including strabismus, optic nerve
atrophy, amblyopia, and cortical visual impairment, are
frequently observed
9,19,22
and, together with rare skeletal
malformations (i.e., scoliosis and fused cervical vertebrae),
constitute corroborating rather than defining features of
LAMSHF syndrome.
9
The incomplete penetrance observed
for some features suggests that SOX5 haploinsufficiency
manifests differently in distinct individual genetic back-
grounds or that some variants retain partial activity. The
investigation of clinical features according to variant types,
however, did not reveal clear genotype–phenotype correla-
tions. Patients with HMG domain missense variants tended to
have milder language deficits, but this finding requires
confirmation with larger patient cohorts. Based on the lack
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WT
a
b
c
d
ef
0
75
150
225
SOX5 wild-type protein
(ng plasmid)
Nonsense variants Missense variants
within the HMG domain
Missense variants
outside the HMG domain
Q208*
Q274*
G354*
R493*
M560V
N561H
R571W
A596P
Y605C
–
C
245
180
135
100
75
63
48
35
200
130.6
100.0
5.3
164.7
113.6
94.7
111.1
92.6
101.4
85.2 92.2 82.3 80.9
170.4
132.5
137.7
100.0
63.7
0.0
4.3 5.9 4.8 2.1 0.0 1.4 0.0 5.0
109.2
73.9
57.1
0.0
Normalized reporter activity
180
160
140
120
100
80
60
40
20
0
200
Normalized reporter activity
180
160
140
120
100
80
60
40
20
0
135
245
180
135
100
75
63
100
75
Wild-type SOX5
DNA SOX5 Membrane Merge DNA SOX5 Membrane Merge DNA SOX5 Membrane Merge
G354* variant N561H variant
245
180
135
100
75
63
48
35
NCNCNCNCNCN CNC N CNCNCNCNCN
Q208* Q274* G354* R493* R235C T632N M560V N561H R571W A596P Y605C
R235C
T632N
0
75
150
–
–
–+– + –+
WT R235C
–WT
WT
WT
M560V
M560V N561H
N561H R571W
R571W
R235W
R235C
T632N
T632N
SOX5
SOX5
dimer
SOX5
monomer
SOX5
Non-sp.
Free
probe
A596P
A596P
Y605C
Y605C
225
300
SOX5 wild-type protein
(ng plasmid)
Nonsense variants Missense variants
within the HMG domain
Missense variants
outside the HMG domain
Q208*
Q274*
G354*
R493*
M560V
N561H
R571W
A596P
Y605C
R235C
T632N
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of obvious genotype–phenotype correlations and on the
observation of variable phenotype severity in unrelated
individuals with identical SOX5 variants, we tentatively
conclude that yet-unidentified factors significantly contribute
to the penetrance and degree of disease severity.
We also describe in this study four patients with de novo
variants located outside the HMG domain and altering amino
acids conserved in SOX5 orthologs. However, the pathogeni-
city of these variants could not be established through
functional assays, and it thus remains unclear whether and
how these variants contribute to disease in these patients.
Three of these patients (V2–V4) had phenotypic features
compatible with LAMSHF syndrome (although patient V2
was very young at the time of the study and patient V3 mainly
had ASD), whereas the fourth patient (V1) had Tourette
syndrome. The variant identified in the latter patient
(Arg235Cys) was also present in four gnomAD individuals
from different ethnicities. Although Tourette patients are
included in gnomAD “neuro”cohorts, the individuals with
Arg235Cys were not in these cohorts, suggesting that these
individuals had no obvious neurological phenotype. Further
investigations are therefore warranted to investigate whether
missense variants outside the HMG domain could impair
untested activities of SOX5 and whether these variants could
predispose to LAMSHF or Tourette syndrome.
In conclusion, our study demonstrates that the genetic and
clinical spectrum in LAMSHF syndrome is much larger than
previously described, and extends to missense variants
clustering in the HMG domain. In silico and in vitro
functional data support the concept that these missense
variants are pathogenic by causing loss of function of the
SOX5 transcription factor, and thereby reflect gene haploin-
sufficiency during neurogenesis and occasionally during other
developmental processes. The impacts of variants located
outside the HMG domain remain to be determined.
SUPPLEMENTARY INFORMATION
The online version of this article (https://doi.org/10.1038/s41436-
019-0657-0) contains supplementary material, which is available
to authorized users.
ACKNOWLEDGEMENTS
We thank the patients and their families for their participation in
this study, and the C4RCD Research Group (Newell Belnap,
Amanda Courtright, Ana Claasen, David Craig, Matt Huentelman,
Madison LaFleur, Sampathkumar Rangasamy, Ryan Richholt,
Isabelle Schrauwen, Ashley L. Siniard, and Szabolics Szelinger) for
providing clinical information on patient P18. This research was
funded in part by the Agence Nationale de la Recherche and
European High-Functioning Autism Network (ANR EUHFAUTISM),
the Assistance Publique–Hôpitaux de Paris (AP-HP), the Institut
National de la Santé et de la Recherche Médicale (INSERM), the
BioPsy labex (to Christel Depienne and C.N.) and the Association
FrançaiseduSyndromeGillesdelaTourette(AFSGT)toChristel
Depienne. It was also funded by the Cleveland Clinic Lerner
Research Institute (LRI Chair’s Innovative Research Award to V.L.),
and by Harper’s Quest and the LAMSHF Syndrome Research Fund
(donations to V.L.) and the Center for Individualized Medicine,
Mayo Clinic. This study makes use of data generated by the
DECIPHER community and the Deciphering Developmental Dis-
orders (DDD) Study, which is funded by the Wellcome Trust. The
DDD study presents independent research commissioned by the
Health Innovation Challenge Fund (grant number HICF-1009-003),
a parallel funding partnership between Wellcome and the
Department of Health, and the Wellcome Sanger Institute (grant
number WT098051). The views expressed in this publication are
those of the author(s) and not necessarily those of Wellcome or the
Department of Health. The study has UK Research Ethics
Committee approval (10/H0305/83, granted by the Cambridge
South REC, and GEN/284/12 granted by the Republic of Ireland
REC). The research team acknowledges the support of the National
Institute for Health Research, through the Comprehensive Clinical
Research Network.
DISCLOSURE
M.J.G.S., K. McWalter, R.E.S., and Z.Z. are employees of GeneDx,
Inc. S.I. is employed by Ambry Genetics, a company that provides
testing for multigene panels and medical exome sequencing. The
Department of Molecular and Human Genetics at Baylor College of
Medicine receives revenue from clinical genetic testing performed at
Baylor Genetics Laboratories. The other authors declare no conflicts
of interest.
Fig. 4 Subcellular localization and activities of SOX5 variants. (a) Western blots of cytoplasmic (C) and nuclear (N) extracts from HEK-293 cells
transfected with plasmids encoding no protein (-), wild-type SOX5 (WT), or SOX5 variants. Blots were incubated with SOX5 antibody. Red boxes, SOX5-
specific protein signals. Numbers, Mr of protein standards. (b) Representative images of SOX5 immunostaining (green signal) in HEK-293 cells transfected
with plasmids encoding wild-type SOX5 (WT) or the indicated variants. Nuclei are seen in blue and plasma membranes in red. Scale bars: 20 μm. (c) Test of the
abilities of SOX5 variants to synergize with SOX9 in transactivation. HEK-293 cells were transfected with Acan and pSV2βGal reporter plasmids and plasmids
encoding no protein, SOX9, and/or SOX5. The WT SOX5 plasmid was used in the indicated amounts, and the variant plasmids at 150 ng. Reporter activities
are presented as the mean ± standard deviation obtained for triplicates in one representative experiment. They were normalized for transfection efficiency and
are reported as increase over the activity of SOX9 alone. (d) Test of the abilities of SOX5 variants to interfere with WT SOX5 in transactivation. HEK-293 cells
were transfected essentially as described above. SOX5 variant plasmids were tested at 150 ng with 150 ng SOX5 WT plasmid. Reporter activities were
calculated and are presented as described above. (e) Test of the abilities of SOX5 variants to bind DNA in electrophoretic mobility shift assay (EMSA). Extracts
from HEK-293 cells transfected with empty, WT SOX5, or SOX5 variant plasmid were incubated with a 2HMG DNA probe. Top, X-ray film images. SOX5/DNA
complexes migrated more slowly than nonspecific protein (non-sp.)/DNA complexes. Bottom, western blot showing similar amounts of all SOX5 proteins. (f)
Dimerization assay with the same extracts as in (c) for no protein, WT SOX5, and the R235C variant. Western blots were performed using SOX5 antibody.
SOX5 dimers ran in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent Mr twice as large as that of monomers.
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ARTICLE ZAWERTON et al
12 Volume 0
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GENETICS in MEDICINE

Ash Zawerton, MS
1
, Cyril Mignot, MD, PhD
2,3
, Ashley Sigafoos, BSc
4
, Patrick R. Blackburn, PhD
5
,
Abdul Haseeb, PhD
6
, Kirsty McWalter, MS, CGC
7
, Shoji Ichikawa, PhD
8
, Caroline Nava, MD, PhD
2,3
,
Boris Keren, MD, PhD
2,3
, Perrine Charles, MD, PhD
3
, Isabelle Marey, MD
3
,
Anne-Claude Tabet, MD, PhD
9,10
, Jonathan Levy, MD, PhD
9
, Laurence Perrin, MD
9
,
Andreas Hartmann, MD
2,11
, Gaetan Lesca, MD, PhD
12,13
, Caroline Schluth-Bolard, MD, PhD
12,13
,
Pauline Monin, MD
12
, Sophie Dupuis-Girod, MD, PhD
12,14
, Maria J. Guillen Sacoto, MD
7
,
Rhonda E. Schnur, MD
7
, Zehua Zhu, PhD
7
, Alice Poisson, MD, PhD
15
, Salima El Chehadeh, MD
16
,
Yves Alembik, MD
16
, Ange-Line Bruel, PhD
17,18
, Daphné Lehalle, MD, PhD
17,19
,
Sophie Nambot, MD
17,19
, Sébastien Moutton, MD
17,19
, Sylvie Odent, MD
20,21
,
Sylvie Jaillard, MD, PhD
22
, Christèle Dubourg, PharmD, PhD
21,23
,
Yvonne Hilhorst-Hofstee, MD, PhD
24
, Tina Barbaro-Dieber, MD
25
, Lucia Ortega, MD
25
,
Elizabeth J. Bhoj, MD, PhD
26
, Diane Masser-Frye, MS, MSW
27
, Lynne M. Bird, MD
27,28
,
Kristin Lindstrom, MD
29
, Keri M. Ramsey, RN
30
, Vinodh Narayanan, MD
30
, Emily Fassi, MS
31
,
Marcia Willing, MD, PhD
31
, Trevor Cole, MBChB FRCP
32
, Claire G. Salter, BMBS, MRCPCH
32,33
,
Rhoda Akilapa, BMBS, MRCPCH
34
, Anthony Vandersteen, MD, PhD
35
, Natalie Canham, MBChB
36,37
,
Patrick Rump, MD, PhD
38
, Erica H. Gerkes, MD
38
, Jolien S. Klein Wassink-Ruiter, MD
38
,
Emilia Bijlsma, MD, PhD
38
, Mariëtte J. V. Hoffer, PhD
24
, Marcelo Vargas, MD
39,40
,
Antonina Wojcik, MS, CGC
39,40
, Florian Cherik, MD
41
, Christine Francannet, MD
41
,
Jill A. Rosenfeld, MS
42
, Keren Machol, MD, PhD
42
, Daryl A. Scott, MD, PhD
42,43
,
Carlos A. Bacino, MD
42
, Xia Wang, PhD
42
, Gary D. Clark, MD
44
, Marta Bertoli, MD
45
,
Simon Zwolinski, PhD
45
, Rhys H. Thomas, MBChB, PhD
46,47
, Ela Akay, MD
47
,
Richard C. Chang, MD
48
, Rebekah Bressi, MS
48
, Rossana Sanchez Russo, MD
49
,
Myriam Srour, MD, PhD
50
, Laura Russell, MD
51
, Anne-Marie E. Goyette, MD
52
, Lucie Dupuis, MS
53
,
Roberto Mendoza-Londono, MD
53
, Catherine Karimov, MD
54
, Maries Joseph, MD
55
,
Mathilde Nizon, MD
56,57
, Benjamin Cogné, PharmD
56,57
, Alma Kuechler, MD
58
,
Amélie Piton, PhD
59,60
, Deciphering Developmental Disorder Study
61
, Eric W. Klee, PhD
5,62
,
Véronique Lefebvre, PhD
6
, Karl J. Clark, PhD
4
and Christel Depienne, PhD
2,58,60
1
Department of Cellular & Molecular Medicine, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA.
2
INSERM U 1127,
CNRS UMR 7225, Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle épinière, ICM, Paris,
France.
3
AP-HP, Hôpital Pitié-Salpêtrière, Département de Génétique et de Cytogénétique; Centre de Référence Déficiences
Intellectuelles de Causes Rares, GRC UPMC « Déficience Intellectuelle et Autisme », Paris, France.
4
Department of Biochemistry and
Molecular Biology, Mayo Clinic, Rochester, MN, USA.
5
Center for Individualized Medicine, Department of Health Science Research,
and Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
6
Department of Surgery, Division of
Orthopaedic Surgery, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA.
7
GeneDx, Gaithersburg, MD, USA.
8
Department of Clinical Diagnostics, Ambry Genetics, Aliso Viejo, CA, USA.
9
Genetics Department, Robert Debré Hospital, APHP,
Paris, France.
10
Human Genetics and Cognitive Functions, Institut Pasteur, Paris, France.
11
APHP, Department of Neurology, Hôpital
de la Pitié-Salpêtrière, Paris, France.
12
Service de Génétique, Hospices Civils de Lyon –GHE, Lyon, France.
13
CNRS UMR 5292, INSERM
U1028, CNRL, and Université Claude Bernard Lyon 1, GHE, Lyon, France.
14
Centre de référence pour la maladie de Rendu-Osler,
Bron, France.
15
GénoPsy, Reference Center for Diagnosis and Management of Genetic Psychiatric Disorders, Centre Hospitalier le
Vinatier and EDR-Psy Team (CNRS & Lyon 1 Claude Bernard University), Lyon, France.
16
Département de Génétique Médicale, CHU
de Hautepierre, Strasbourg, France.
17
INSERM 1231 LNC, Génétique des Anomalies du Développement, Université de Bourgogne-
Franche Comté, Dijon, France.
18
FHU-TRANSLAD, Université de Bourgogne/CHU Dijon, Dijon, France.
19
Centre de Génétique et
Centre de Référence Maladies Rares «Anomalies du Développement de l’Interrégion Est», Hôpital d’Enfants, CHU Dijon Bourgogne,
Dijon, France.
20
CHU de Rennes, service de génétique clinique, Rennes, France.
21
Univ Rennes, CNRS, IGDR, UMR 6290, Rennes,
France.
22
Univ Rennes, CHU Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085,
Rennes, France.
23
Service de Génétique Moléculaire et Génomique, CHU, Rennes, France.
24
Department of Clinical Genetics, Leiden
University Medical Center, Leiden, Netherlands.
25
Cook Childrens Medical Center, Fort Worth, TX, USA.
26
Department of Clinical
Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA.
27
Rady Children’s Hospital San Diego, Division of Genetics and
Dysmorphology, San Diego, CA, USA.
28
Department of Pediatrics, University of California–San Diego, San Diego, CA, USA.
29
Division of Genetics and Metabolism, Phoenix Children’s Hospital, Phoenix, AZ, USA.
30
Translational Genomics Research Institute
(TGen), Center for Rare Childhood Disorders, Phoenix, AZ, USA.
31
Division of Genetics and Genomic Medicine, Department of
Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.
32
West Midlands Regional Genetics Service and
Birmingham Health Partners, Birmingham Women’s and Children’s NHS Foundation Trust, Birmingham, UK.
33
RILD Wellcome
Wolfson Centre, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.
34
North West Thames Regional Genetics Service,
Northwick Park Hospital, Harrow, London, UK.
35
IWK Health Centre, Dalhousie University, Halifax, NS, Canada.
36
North West
Thames Regional Genetics Service, Northwick Park Hospital, London, UK.
37
Cheshire & Merseyside Regional Genetics Service,
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Liverpool Women’s Hospital, Liverpool, UK.
38
Department of Genetics, University of Groningen, University Medical Center
Groningen, Groningen, Netherlands.
39
Gillette Children’s Specialty Healthcare, St. Paul, MN, USA.
40
Children’s Minnesota,
Minneapolis, MN, USA.
41
Service de génétique clinique, Centre de Référence Maladies Rares «Anomalies du Développement et
syndromes malformatifs du Sud-Est”, CHU de Clermont-Ferrand, Clermont-Ferrand, France.
42
Department of Molecular & Human
Genetics, Baylor College of Medicine, Houston, TX, USA.
43
Department of Molecular Physiology and Biophysics, Baylor College of
Medicine, Houston, TX, USA.
44
Pediatrics–Neurology, Baylor College of Medicine, Houston, TX, USA.
45
Northern Genetics Service—
Newcastle upon Tyne NHS Foundation Trust, Newcastle upon Tyne, UK.
46
Institute of Neuroscience, Newcastle University,
Framlington Place, Newcastle upon Tyne, UK.
47
Department of Neurology, Royal Victoria Infirmary, Newcastle upon Tyne Hospitals
NHS Foundation Trust, Newcastle upon Tyne, UK.
48
Division of Metabolic Disorders, Children’s Hospital of Orange County (CHOC),
Orange, CA, USA.
49
Department of Human Genetics, Emory Universit, Atlanta, GA, USA.
50
Division of Pediatric Neurology,
Department of Pediatrics, Montreal Children’s Hospital, McGill University Health Center, Montreal, QC, Canada.
51
Division of
Medical Genetics, Department of Specialized Medicine, McGill University, Montreal, QC, Canada.
52
Child Development Program,
Department of Pediatrics, Montreal Children’s Hospital, McGill University Health Center, Montreal, QC, Canada.
53
Division of
Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto, Toronto, ON, Canada.
54
Children’s
hospital of Los Angeles, Los Angeles, CA, USA.
55
Medical Genetics and Metabolism, Valley Children’s Hospital, Madera, CA, USA.
56
CHU Nantes, Service de Génétique Médicale, Nantes, France.
57
INSERM, CNRS, UNIV Nantes, l’institut du thorax, Nantes, France.
58
Institut für Humangenetik, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany.
59
Laboratoire de Diagnostic
Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
60
IGBMC, CNRS UMR 7104/INSERM U964/Université de
Strasbourg, Illkirch, France.
61
DDD Study, Wellcome Sanger Institute, Hinxton, Cambridge, UK.
62
Department of Clinical Genomics,
Mayo Clinic, Rochester, MN, USA
ARTICLE ZAWERTON et al
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GENETICS in MEDICINE